Morphology Methods: Cell and Molecular Biology Techniques

MORPHOLOGY METHODS Cell and Molecular Biology Techniques Edited by

RICARDO V. LLOYD, MD, PhD

HUMANA PRESS

Morphology Methods

MORPHOLOGY METHODS Cell and Molecular Biology Techniques Edited by

Ricardo V. Lloyd, MD, PhD Mayo Clinic, Rochester, MN

Foreword by

Ronald A. DeLellis, MD Weill Medical College of Cornell University and New York Presbyterian Hospital, New York, NY

Humana P ress Totowa, New Jersey

© 2001 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 www.humanapress.com

All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. The content and opinions expressed in this book are the sole work of the authors and editors, who have warranted due diligence in the creation and issuance of their work. The publisher, editors, and authors are not responsible for errors or omissions or for any consequences arising from the information or opinions presented in this book and make no warranty, express or implied, with respect to its contents. This publication is printed on acid-free paper.

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ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials. Cover illustrations: Backround is figure 18 and inset is figure 5 from Chapter 4, “In Situ Detection of Infectious Agents,” by Gary W. Procop and Randall Hayden. Cover design by Patricia F. Cleary. For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel: 973-256-1699; Fax: 973-256-8341; E-mail: [email protected], or visit our Website: http://humanapress.com Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $10.00 per copy, plus US $00.25 per page, is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [0-89603-955-2/01 $10.00 + $00.25]. Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 Library of Congress Cataloging-in-Publication Data Morphology methods : cell and molecular biology techniques / edited by Ricardo V. Lloyd. p. cm. Includes bibliographical references and index. ISGN 0-89603-955-2 (alk. paper) 1. Cytology--Technique. 2. Molecular biology--Technique. 3. Fluorescence in situ hybridization. 4. Polymerase chain reacion. 5. Immuniohitochemistry. I. Lloyd, Ricardo V. QH585 .M67 2001 571.6'028--dc21 2001016959

Foreword

The past several decades have witnessed an impressive array of conceptual and technological advances in the biomedical sciences. Much of the progress in this area has developed directly as a result of new morphology-based methods that have permitted the assessment of chemical, enzymatic, immunological, and molecular parameters at the cellular and tissue levels. Additional novel approaches including laser capture microdissection have also emerged for the acquisition of homogeneous cell populations for molecular analyses. These methodologies have literally reshaped the approaches to fundamental biological questions and have also had a major impact in the area of diagnostic pathology. Much of the groundwork for the development of morphological methods was established in the early part of the 19th century by Francois-Vincent Raspail, generally acknowledged as the founder of the science of histochemistry. The earliest work in the field was primarily in the hands of botanists and many of the approaches to the understanding of the chemical composition of cells and tissues involved techniques such as microincineration, which destroyed structural integrity. The development of aniline dyes in the early 20th century served as a major impetus to studies of the structural rather than chemical composition of tissue. Later in the century, however, the focus returned to the identification of chemical constituents in the context of intact cell and tissue structure. Ultimately, it became possible to localize with great precision major classes of nucleic acids, proteins, and individual amino acids, lipids, and carbohydrates on the basis of specific chemical reactions that had been adapted for histological and cytological preparations. The development of the immunofluorescence technique for the localization of pneumococcal antigens in 1942 by Albert Coons and his colleagues provided the foundation for the subsequent development of enzyme-based immunohistochemical procedures. The advantages of these methods were related to the facts that the reaction products could be visualized directly in the light microscope and that the multistep staining procedures possessed sufficient sensitivity to localize even the small amounts of antigen that survived formalin fixation and paraffin embedding. The subsequent development of increasingly more sensitive immunohistochemical staining sequences and the availability of increasing numbers of monoclonal antibodies together with the development of antigen retrieval technology has made immunohistochemistry clearly one of the most important research and diagnostic tools in the armamentarium of the investigator.

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The development of molecular technologies has had an equally profound impact on approaches to the analysis of basic biological questions directly in tissue sections and single cells. In situ hybridization techniques, including FISH, have emerged as powerful techniques for the localization of DNA and RNA sequences at the cellular and subcellular levels. Increased sensitivity of in situ hybridization has been made possible both by amplification of target sequences through the use of the polymerase chain reaction (PCR) and in situ reverse transcriptase (RT)—PCR methods, as well as by approaches to enhance signal amplification, including the use of increasingly more sensitive nonradioactive techniques. Tissue and cDNA microarrays have provided a particularly useful approach for large scale analyses of DNA, RNA, or protein and for studies of gene expression. The major objective of Dr. Lloyd’s book is to present an overview of the major areas relevant to the current practice of molecular morphology. Both he and his distinguished group of contributors have been particularly successful in achieving this goal and in providing succinct overviews of specific methodologies, their major applications and detailed protocols for their performance. Dr. Lloyd’s monograph will undoubtedly find an important place on the bookshelves of basic scientists and clinical investigators who have an interest in correlating morphological and molecular parameters. Ronald A. DeLellis, MD Professor of Pathology Weill Medical College of Cornell University Vice Chair for Anatomic Pathology New York Presbyterian Hospital New York, NY

Preface

Molecular morphologic methods—defined as molecular and cell biologic analyses of tissues in which the architectural integrity and spatial interrelationship of the cells being studied are preserved—have increased rapidly in number and versatility during the past few years. These changes have occurred both in diagnostic pathology and in basic scientific research. Several ongoing developments affecting the pathology and the scientific communities should make this book a valuable resource. First, it is usually difficult for pathologists and investigators interested in molecular morphology to learn rapidly from a single source about methods suitable to specific diagnostic and experimental questions. Second, the completion of the human genome project in the near future will provide the foundation to learn about the functions of myriad of genes with unique roles in specific cells and tissues, so a morphologic basis for the study of human genes and understanding human diseases will be in greater demand. There is no good single source available that discusses in detail the most significant aspects of recent cell biologic techniques by outstanding experts in their fields. Such a book is needed to keep up with scientific research in morphology and recent pathologic diagnostic techniques relevant in the twenty-first century. Our objective was to produce a book addressing the major areas relevant to molecular morphology today. Many of the chapters include detailed protocols for setting up or performing techniques now in use. Potential pitfalls and anticipated problems are also discussed. Practicing pathologists interested in recent developments and researchers interested in molecular morphology for designing experiments, for teaching undergraduate, graduate, and professional students, or simply for keeping up with the literature detailing molecular morphologic approaches—all will find here the technical and scientific background to accomplish their objectives. The publication of Morphology Methods: Cell and Molecular Biology Techniques would not have been possible without the enthusiastic support and contributions of Mr. Thomas Lanigan, President, and Mr. John Morgan of Humana Press. Ricardo V. Lloyd, MD, PhD

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Contents

Foreword ................................................................................................................. v Preface .................................................................................................................. vii Contributors ........................................................................................................ xiii Color Plates .......................................................................................................... xv 1 Introduction to Molecular Methods Ricardo V. Lloyd .................................................................................................... 1 2 Laser Capture Microdissection: Principles and Applications Ricardo V. Lloyd .................................................................................................. 11 PROTOCOL: H&E Staining

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PROTOCOL: Operating the PIX Cell System

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PROTOCOL: DNA Extraction from Collected Cells 24 PROTOCOL: RNA Extraction from Collected Cells 25

3 In Situ Hybridization: Detection of DNA and RNA Long Jin, Xiang Qian, and Ricardo V. Lloyd ................................................... 27 PROTOCOL: In Situ Hybridization

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4 In Situ Detection of Infectious Agents Gary W. Procop and Randall Hayden ............................................................... 47 PROTOCOL: Detecting Adenovirus 61 PROTOCOL: Epstein-Barr Virus (Eber I/II)

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5 Fluorescent In Situ Hybridization (FISH) Susan Sheldon ..................................................................................................... 67 PROTOCOL: FISH Materials

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6 Practical Applications of the FISH Technique George Kontogeorgos, Nikiforos Kapranos, and Eleni Thodou ..................... 91 PROTOCOL: Metaphase Analysis from Whole Blood Culture PROTOCOL: Metaphase Analysis from Solid Tumors 106

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PROTOCOL: Interphase Analysis on Cell Imprints—Cytology Specimens 108 PROTOCOL: Interphase Analysis of Formalin-Fixed Paraffin-Embedded Tissues 110

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Contents 7 Tyramide Amplification Methods for In Situ Hybridization John L. Frater and Raymond R. Tubbs .......................................................... 113 PROTOCOL: TSA by Rapp et al., 1995 PROTOCOL: CARD/TSA with Nanogold

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PROTOCOL: Tyramide-enhanced FISH with Peroxidase-labeled Oligonucleotides 127

8 PROTOCOLS: Ultrastructural In Situ Hybridization Akira Matsuno, Tadashi Nagashima, R. Yoshiyuki Osamura, and Keiichi Watanabe................................................................................... 129 9 Quantitation of In Situ Hybridization Analysis Lars-Inge Larsson ............................................................................................. 145 10 PROTOCOLS: Confocal Laser Scanning Microscopy Akira Matsuno, Johbu Itoh, Tadashi Nagashima, R. Yoshiyuki Osamura, and Keiichi Watanabe .......................................... 165 11 PROTOCOLS: Polymerase Chain Reaction Yuri E. Nikiforov and Philip N. Howles.......................................................... 181 12 PROTOCOLS: Detection of Nucleic Acids in Cells and Tissues by In Situ Polymerase Chain Reaction Omar Bagasra, Lisa E. Bobroski, and Muhammad Amjad ........................... 209 13 Immunohistochemistry: Theory and Practice Patrick C. Roche and Eric D. Hsi .................................................................... 229 14 Antigen Retrieval Technique for Immunohistochemistry: Principles, Protocols, and Further Development Clive R. Taylor and Shan-Rong Shi ................................................................ 239 PROTOCOL: Antigen Retrieval (AR) Technique

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15 Tyramide Amplification in Immunohistochemistry Naoko Sanno, Akira Teramoto, and R. Yoshiyuki Osamura ........................ 267 PROTOCOL: CARD Immunohistochemistry

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16 Application of Immunohistochemistry in the Diagnosis of Lymphoid Lesions Paul J. Kurtin .................................................................................................... 277 17 Applications of Immunohistochemistry in the Diagnosis of Undifferentiated Tumors Mark R. Wick and Lisa A. Cerilli .................................................................... 323

Contents

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18 Applications of Immunohistochemistry in the Diagnosis of Endocrine Lesions Ricardo V. Lloyd ................................................................................................ 361 19 Ultrastructural Immunohistochemistry Sergio Vidal, Eva Horvath, and Kalman Kovacs ........................................... 375 PROTOCOL: Ultrastructural Immunohistochemistry

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20 PROTOCOLS: Clonality Analysis Aurel Perren and Paul Komminoth ................................................................. 403 Index ........................................................................................................................... 413

Contributors

MUHAMMAD AMJAD, PhD • Laboratories of Transgenic and Recombinant Vaccine, Department of Biology, Lincoln University, Lincoln University, PA OMAR BAGASRA, MD, PhD • Laboratories of Transgenic and Recombinant Vaccine, Department of Biology, Lincoln University, Lincoln University, PA LISA E. BOBROSKI, MS • Laboratories of Transgenic and Recombinant Vaccine, Department of Biology, Lincoln University, Lincoln University, PA LISA A. CERILLI, MD • Fechner Laboratory of Surgical Pathology, University of Virginia Medical Center, Charlottesville, VA JOHN L. FRATER, MD • Department of Clinical Pathology, Cleveland Clinic Foundation, Cleveland, OH RANDALL HAYDEN, MD • Clinical and Molecular Microbiology, St. Jude Children's Research Hospital, Cleveland, OH EVA HORVATH, PhD • Department of Laboratory Medicine, St. Michael’s Hospital, University of Toronto, Toronto, Ontario, Canada PHILIP N. HOWLES, PhD• Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, OH ERIC D. HSI, MD • Departments of Anatomic and Clinical Pathology, Cleveland Clinic Foundation, Cleveland, OH JOHBU ITOH, MD, PhD • Laboratories for Structure and Function Research, Tokai University School of Medicine, Boseidai, Isehara City, Kanagawa, Japan NIKIFOROS KAPRANOS, MD, PhD • Department of Pathology, Molecular Division, Amalia Fleming Hospital, Athens, Greece PAUL KOMMINOTH, MD • Department of Pathology, Hospital Baden, Baden, Switzerland GEORGE KONTOGEORGOS, MD, PhD • Department of Pathology, G. Gennimatas General Hospital of Athens, Athens, Greece KALMAN KOVACS, MD, PhD • Department of Laboratory Medicine, St. Michael’s Hospital, University of Toronto, Toronto, Ontario, Canada PAUL J. KURTIN, MD • Divisions of Anatomic Pathology and Hematopathology, Mayo Clinic, Rochester, MN LARS-INGE LARSSON, DSc • Division of Cell Biology, Department of Anatomy and Physiology, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark RICARDO V. LLOYD, MD, PhD • Department of Laboratory Medicine and Pathology, Mayo Clinic and Foundation, Rochester, MN xiii

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LONG JIN • Mayo Clinic and Foundation, Rochester, MN AKIRA MATSUNO, MD, PhD • Department of Neurosurgery, Teikyo University Ichihara Hospital, Ichihara City, Chiba, Japan TADASHI NAGASHIMA, MD, PhD • Department of Neurosurgery, Teikyo University Ichihara Hospital, Ichihara City, Chiba, Japan YURI E. NIKIFOROV, MD, PhD • Department of Pathology and Laboratory Medicine, Univeristy of Cincinnati College of Medicine, Cincinnati, OH R. YOSHIYUKI OSAMURA, MD, PhD • Department of Pathology, Tokai University School of Medicine, Boseidai, Isehara City, Kanagawa, Japan AUREL PERREN, MD • Department of Pathology, University Hospital Zürich, Zürich, Switzerland GARY W. PROCOP, MD • Clinical Microbiology, Cleveland Clinic Foundation, Cleveland, OH XIANG QIAN • Mayo Clinic and Foundation, Rochester, MN PATRICK C. ROCHE, PhD • Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN NAOKO SANNO, MD, PhD • Department of Neurosurgery, Nippon Medical School, Tokyo, Japan SUSAN SHELDON, PhD • Department of Pathology, University of Michigan, Ann Arbor, MI SHAN-RONG SHI, MD • Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA CLIVE R. TAYLOR, MD, PhD • Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA AKIRA TERAMOTO, MD, DMSc • Department of Neurosurgery, Nippon Medical School, Tokyo, Japan ELENI THODOU, MD, PhD • Department of Pathology, G. Gennimatas General Hospital of Athens, Athens, Greece RAYMOND R. TUBBS, DO • Department of Clinical Pathology, Cleveland Clinic Foundation, Cleveland, OH SERGIO VIDAL, DVM, PhD • Department of Laboratory Medicine, St. Michael’s Hospital, University of Toronto, Toronto, Ontario, Canada; Laboratory of Histology, University of Santiago de Compostela, Lugo, Spain KEIICHI WATANABE, MD, PhD • Department of Pathology, Tokai University School of Medicine, Boseidai, Isehara City, Kanagawa, Japan MARK R. WICK, MD • Fechner Laboratory of Surgical Pathology, University of Virginia Medical Center, Charlottesville, VA

Color Plates

Color Plates 1–8 follow page 208. Color Plate 1 Fig. 1, Chapter 3. (A) In situ hybridization detects EBV in a posttransplant lymphoproliferative disorder. (B) In situ hybridization to detect EBV in a nasopharyngeal carcinoma metastatic to a cervical lymph node. (C) Detection of κ (left) and λ (right) in a plasmacytic lymphoma. (D) In situ hybridization for albumin to diagnose a hepatocellular carcinoma metastatic to the scapula. (E) Chromogranin A and B expression in the adrenal cortex. (F) Pro-insulin mRNA expression in the normal pancreatic islets. See discussion on pages 36, 39 and full caption on page 37. Color Plate 2 Figs. 1–19, Chapter 4. See pages 47–55. • Fig. 1. Lymph noted with EBV, ISH. • Fig. 2. Pharyngeal biopsy with HPV, ISH. • Fig. 3. Placenta, involved by CMV, ISH. • Fig. 4. Oral cavity (palate) biopsy with HSV, H&E. • Fig. 5. Palate biopsy with HSV (Fig. 4) confirmed by ISH. • Fig. 6. Lung tissue with adenovirus, H&E. • Fig. 7. Adenovirus in lung tissue (Fig. 6) confirmed by ISH. • Fig. 8. Brain biopsy showing JC viral inclusion in oligodendrocytes, H&E. • Fig. 9. JC virus in brain biopsy (Fig. 8) confirmed by ISH. • Fig. 10. Cryptococcus neoformans in an open lung biopsy, GMS. • Fig. 11. Identification of the yeast forms in Fig. 10 as C. neoformans using ISH. • Fig. 12. Histoplasma capsulatum in an open lung biopsy, GMS. • Fig. 13. Confirmation of the yeast forms in Fig. 12 as H. capsulatum using ISH. • Fig. 14. Open lung biopsy with Blastomyces dermatiditis, GMS. • Fig. 15. Confirmation of the yeast forms in Fig. 14 with ISH. • Fig. 16. Endospores and immature spherules of Coccidiodes immitis in a lung biopsy, GMS. • Fig. 17. Confirmation of C. immitis in Fig. 16 by ISH. • Fig. 18. Open lung biopsy showing Legionella pneumophila, Warthin-Starry stain. • Fig. 19. Identification of L. pneumophila in Fig. 18 by ISH. Color Plate 3 Figs. 5, 6, and 7, Chapter 5. See pages 74–79 for discussion and additional details. • Fig. 5. Two-color microdeletion probe. • Fig. 6. A two-color probe for BCR and ABL genes on a touch preparation from a spleen. • Fig. 7. Painting probe for chromosome 9. Color Plate 4 Fig. 3, Chapter 6. See pages 94–95. (A) Examples of numerical chromosome aberrations using an α-satellite, centromere-specific DNA probe. (B) Monosomy of chromosome 11 as the dominant abnormality in a mixed somatotrophlactotroph, mostly prolactin-producing pituitary adenoma (FITC/PI). (C) Chromosome 1 in ductal carcinoma of the breast. (D) Chromosome 2 breast carcinoma of the ductal type. (E) Localization of the DiGeorge gene on a chromosome in the 22q11 region in normal metaphase spreads. (F) Dualcolor FISH in metaphases of normal lymphocytes demonstrates the bcr gene on chromosome 22 (green) and the abl gene on chromosome 9 (red) (FITC/TR/DAPI). (G) Mixture of whole paint (coatsome) DNA probe for chromosome 11 (red) and α-satellite centromere-specific probe or chromosome 2 (green) in normal metaphase spreads (FITC/TR/DAPI). (H) All human telomeres demonstrate all chromosomes in metaphase spreads (FITC/PI).

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Color Plate 5 Figs. 9 and 10, Chapter 10. • Fig. 9. Three-dimensional projection images of ACTH and GH cells in normal rat pituitary. (A) ACTH cells are closely associated with the microvessel networks. (B) ACTH signals show a granular pattern in the cytoplasm. (C) GH cells are clearly visible three-dimensionally by CLSM reflectance mode. (D) GH signals appear granular in the cytoplasm three-dimensionally by CLSM reflectance mode. See discussion on page 174 and full caption on page 176. • Fig. 10. Three-dimensional reconstructed images of bilaterally adrenalectomized and ACTH administered rat pituitary glands. See discussion on pages 174–175 and full caption on page 177. Color Plate 6 Figs. 1–5, Chapter 16. • Fig. 1. Immunoperoxidase stains for κ (A) and λ (B) immunoglobulin light chains on frozen section of a case of B-cell small lymphocytic lymphoma. See discussion and full caption on pages 280, 281. • Fig. 2. Immunoperoxidase stains for κ (A) and λ (B) immunoglobulin light chains on paraffin sections of a case of mantle cell lymphoma. See discussion on page 280 and full caption on page 282.• Fig. 3. B-cell small lymphocytic lymphoma. H&E section demonstrating the typical cytologic features (A). Frozen section immunoperoxidase stains for CD20 (B), CD5 (C), CD3 (D), and CD23 (E). See discussion on page 283 and full caption on page 284. • Fig. 4. Angioimmunoblastic T-cell lymphoma stained in frozen sections for CD2 (A) and for CD7 (B). See discussion on page 286 and full caption on page 287. • Fig. 5. Follicular lymphoma stained for bcl-2 paraffin sections. See discussion on page 288 and full caption on page 289. Color Plate 7 Figs. 6–13, Chapter 16. • Fig. 6. Mantle cell lymphoma stained for cyclin D1 in paraffin sections. See discussion and full caption on pages 289, 290. • Fig. 7. CD30-positive anaplastic large cell lymphoma stained for p80 paraffin sections. See discussion and full caption on pages 290, 291. • Fig. 8. Diffuse large B-cell lymphoma involving a lymph node. H&E section (A) and immunoperoxidase stain (B). See discussion and full caption on pages 292, 293. • Fig. 9. Angioimmunoblastic T-cell lymphoma involving a lymph node. H&E section (A) and immunoperoxidase stain (B) for CD3 performed on a paraffin section of the tumor. See discussion and full caption on pages 294, 295. • Fig. 10. Immunoperoxidase stain for CD10 performed on a paraffin section of a follicular lymphoma. See discussion and full caption on pages 296, 297. • Fig. 11. Immunoperoxidase stain for CD23 performed on a paraffin section of an angioimmunoblastic T-cell lymphoma. Same case as Fig. 9. See discussion and full caption on pages 298, 299. • Fig. 12. Immunoperoxidase stain performed on a paraffin section for the cytolytic granule protein Tia-1 in a case of NK/T-cell lymphoma of the nasal type. See discussion and full caption on pages 298, 299. • Fig. 13. Immunoperoxidase stain for CD30 performed on a CD30-positive anaplastic large cell lymphoma. See full caption and discussion on pages 300, 301. Color Plate 8 Figs. 14–19, Chapter 16 • Fig. 14. Hodgkin's lymphoma, nodular sclerosis type involving a lymph node. H&E section demonstrating the large neoplastic cells admixed with small lymphocytes, eosinophils, plasma cells, and macrophages (A). Immunoperoxidase stains for CD15 (B), CD30 (C), and fascin (D) performed on paraffin sections. See discussion and full caption on pages 301–303. • Fig. 15. Hodgkin's lymphoma, nodular lymphocyte predominance type, involving a lymph node. See discussion on pages 302, 304, and full caption on page 305. • Fig. 16. Acute lymphoblastic leukemia, B-lymphocyte precursor type. See discussion and full caption on pages 305, 306. • Fig. 17. Acute myelogenous leukemia, FAB subtype M1. See discussion and full caption on pages 306, 307. • Fig. 18. Langerhans' cell histiocytosis involving the parotid gland. H&E (A) exhibits the typical cytologic features of this disorder. Immunoperoxidase stain for CD1a (B) performed in paraffin sections. See discussion and full caption on pages 307, 308. • Fig. 19. Histiocytic sarcoma. Immunoperoxidase stains for CD45 (A) and CD68 (B), in paraffin sections, and for CD13 on frozen sections of the tumor (C). See discussion on page 308 and full caption on page 309.

1 Introduction to Molecular Methods Ricardo V. Lloyd, MD, PHD GENE STRUCTURE The human genome contains 23 pairs of chromosomes with 3.5 × 109 nucleotide base pairs (bp) in length. With the near completion of the sequencing of the human gene, the best estimate of functional genes appears to be between 30,000 and 40,000 (1–3). Approximately 90% of the human DNA has no known coding function, but a significant amount of DNA, especially in regions close to functional genes, has regulatory roles in cells. However, most of the functions of much of the DNA remain unknown. Specific restriction enzymes derived from prokaryotes are used to cleave DNA at specific sequences, which helps in the analysis of the large amount of genomic DNA. For example, EcoRI, which is derived from Escherichia coli, cleaves DNA at the G↓ AATTC sequence. Because of the specificity of restriction endonucleases, the DNA can be manipulated by genetic engineering, since DNA from any source cut by EcoRI will produce identical ends that hybridize to any other source of DNA produced by digestion with these restriction enzyme (4,5). CHEMICAL COMPOSITION OF NUCLEIC ACIDS DNA and RNA are made of nucleotides, sugars, and phosphate groups (Figs. 1–3). DNA has four bases, the purines adenine and guanine and the pyrimidines cytosine and thymine. In RNA molecules, uracil is substituted for cytosine. The bases are linked to sugar groups (nucleoside), which are in turn linked to phosphates (nucleotides). DNA and RNA are polynucleotide molecules with alternating series of sugar and phosphate groups (Fig. 1). The nucleotide sequences of a polynucleotide is usually written in the 5′ to 3′ order. The nucleotide sequence of DNA contains the genetic information. The four DNA polynucleotide structures are base-paired in a complementary and parallel manner (Fig. 2). One strand runs from 3′ to 5′, and the other strand runs from 5′ to 3′. The DNA structure is twisted in a helix with one complete turn every three base pairs. During hybridization, which is one of the basic tools in molecular analyses, the two strands anneal or come together by base-pairing. Humans somatic cells contain 46 chromosomes, which are made up of 23 pairs. The 23 pairs represent the haploid number of chromosomes, and the 46 chromosomes

From: Morphology Methods: Cell and Molecular Biology Techniques Edited by: R. V. Lloyd  Humana Press, Totowa, NJ

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Fig. 1. Structure of molecules making up DNA and RNA. The bases (cytosine, guanine, thymine, adenine, and unacil) are linked via hydrogen bonds. (A) The purines are linked by three pairs of hydrogen bonds. (B) The pyrimidines are linked by two pairs of hydrogen bonds. (C) The structure of unacil, present in RNA in place of thymine. (D) and (E) Structures of the pentose sugars in DNA and RNA.

represent the diploid number, which represents the two homologous sites of each chromosome. The sex chromosomes are different in males and females; females have two X chromosomes, and males have a Y and an X chromosome. The karyotype represents the complete chromosomal complement of a cell. These karyotypes are usually analyzed by cytogeneticists. Chromosomes are linked by a centromere with two sister chromatid portions. Chromosome maps are based on the position of the banding pattern (easily visualized after staining with specific dyes) and the position of the centromeres. Chromosomes are numbered as 1–22 plus the sex chromosomes. The short arm of the chromosomes is designated as a p and the long arm as q. The p and q arms are divided into various sections. Thus a designation of 5q21 would represent the long arm of chromosome 5, section 2, and band 1 (Fig. 4). RNA AND PROTEIN SYNTHESIS Transcription is the process by which RNA is synthesized from DNA (Fig. 5). The RNA is complementary to the original DNA strand. RNA is synthesized 5′–3′ by RNA polymerases. There are different forms of RNA. For example, transfer RNA, small nuclear RNA, and ribosomal RNA are end products of transcription. In contrast, messenger RNA (mRNA) constitutes most of the RNA that encodes specific protein products

Introduction to Molecular Methods

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Fig. 2. Structure of double-stranded DNA. The deoxyribonucleotide residues (black areas) of each single strand of DNA are linked by phosphodiester bonds between the 3′ carbon of the first deoxyribose residue and the 5′ carbon of the next residue. The purines and pyrimidines are linked by hydrogen bonds. The two DNA strands are on antiparallel orientations.

Fig. 3. Structure of the double helix, showing the three-dimensional relationship among the bases, sugars, and phosphodiester moieties.

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Fig. 4. Physical map of chromosome 7 after Giemsa staining and G-banding. Giemsa staining highlights the dark and light bands. The centromere and sister chromatids are shown.

Fig. 5. Diagrammatic representation of transcription and translation processes leading to specific protein synthesis. The regulatory sequence with the enhancer and promoter sequences are located in the 5′ upstream region. Enhancers bind protein factors and help to determine the rate of transcription. The coding sequences downstream include exons and introns. Introns are spliced out of the mature RNA. AUG is the start codon for protein coding, and there are three stop codons to terminate protein synthesis (UAA, UAG, and UGA). The 5′ CAP site is at the 5′ end, and the poly(A) tail is present at the 3′ end. These sites help to increase the stability of the mature mRNA and optimize protein synthesis.

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Fig. 6. The genetic code, with the amino acid symbols at the top and codons with the amino acids they specify at the bottom.

by translation. The RNA in the nucleus is processed by excision of the intron regions and addition of the 5′CAP site and a 3′ poly(A) tail, which increases the stability of the mature mRNA. The introns that do not encode proteins are also removed from the mRNA. The small nuclear RNA assists in the processing of mRNA. The mature mRNA is transported to the cytoplasm, where it is then translated to protein. The nucleotide sequence of the RNA (and parent DNA) determines the amino acid sequence of the newly synthesized proteins. The genetic code (Fig. 6) which consists of specific codons, specifies one or more amino acids that are essential for protein synthesis. Because there are four bases in RNA and 43 (or 64) possible base triplets or codons, the 20 naturally occurring amino acids can be specified by different codons (Fig. 6). The methionine codon (AUG) is the start codon for protein synthesis, and UAA, UAG, and UGA are the stop codons. mRNA translation begins at the 5′ to 3′ end and is carried out on the ribosomes with the assistance of transfer RNA (tRNA). Codons are recognized by tRNA that have an anticodon triplet complement. The tRNA molecules associate with specific amino acids determined by the anticodon sequence. The tRNA then interacts with the complimentary codon on a message that is being translated, and the ribosome catalyzes the addition of the amino acid to the protein during chain elongation. HYBRIDIZATION Hybridization or annealing involves pairing of complementary strands of nucleic acids. Hybrids can be formed between complementary strands of nucleic acids (DNA

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Fig. 7. Schematic of Southern hybridization. After the genomic DNA is digested by specific restriction endonucleases, the DNA fragments are separated by gel electrophoresis and then transferred to a nylon membrane and denatured with alkali. The single-stranded DNA is hybridized with a labeled probe. Hybrids are formed between the probe and complementary DNA fragments and can be detected with a radioactive or nonradioactive reporter system. Et br, ethidium bromide.

and RNA). These hybrid strands can be reversibly separated by heat treatment or alkaline treatment to disrupt the hydrogen bonds between complementary bases. Hybridization is one of the more powerful tools in molecular analysis. If a tag or label is placed on one strand of the nucleic acid, it can be used as a probe in the analysis of the nucleic acids. These types of analyses are designated as Southern and Northern hybridization for DNA and RNA analyses, respectively, and in situ hybridization for RNA or DNA tissue analysis (Figs. 7–9). In the typical Southern or Northern hybridization, the nucleic acids are separated on a gel by electrophoresis. Because nucleic acids are negatively charged, they move to the positive electrode. After denaturation and/or transfer to a solid matrix, they can be hybridized with a known probe to detect the DNA or RNA of interest. The specificity of the hybridization can be readily controlled by the stringency of the conditions, i.e., pH, temperature, and salt concentration. High stringency can lead to detection of small differences or changes such as a change in a single base, as in a genetic mutation. Hybridization analyses are used in molecular diagnostics to detect gene mutations or infectious agents in body fluids or tissues or to study gene expression in tumors or other diseased tissues. SINGLE-STRAND CONFORMATION POLYMORPHISM ANALYSIS The single-strand conformation polymorphism (SSCP) technique is a useful method for detecting gene mutations using fresh tissues or paraffin sections (6–8). One can

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Fig. 8. Northern hybridization. Total or poly(A)+RNA is separated into fragments of different sizes by gel electrophoresis. Ethidium bromide (Et br) staining allows visualization of the fragments (S) as well as the 28s and 18s ribosomal RNA bands under fluorescence. After it is transferred to a nylon membrane and hybridized with a labeled probe, the mRNA of interest can be detected by autoradiography or with nonradioactive methods. A−A, amino acids.

Fig. 9. In situ hybridization. The probe (DNA or RNA) is labeled with a radioactive or nonradioactive detection system. The DNA probe is hybridized with the messenger RNA associated with the rough endoplasmic reticulum. After hybridization, the target cells or tissues of interest can be detected by autoradiography or with nonisotopic methods such as with a digoxigenin-labeled probe. Advantages of in situ hybridization include more precise intracellular localization of the target and direct visualization of the positive cell relative to the adjacent cells or tissues.

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Fig. 10. Strategy for performing single-strand conformation polymorphism (SSCP) analysis and sequence analysis from paraffin-embedded tissue sections. The polymerase chain reaction (PCR) products can be visualized on a gel and then used for sequencing. SSCP also can be performed with labeled probes and then analyzed on a polyacrylamide gel to detect differences in migration of the single-stranded DNA compared with a known wild-type DNA. The gene fragments with a shift in the migration pattern can be analyzed by sequencing to detect the gene mutation or other genetic changes.

readily detect sequence changes in a single-stranded DNA fragment. Single-stranded DNA fragments with a unique polyacrylamide gel in a manner dependent on the folded conformation, which is in turn dependent on ionic strength and temperature. The fragments are usually between 50 and 400 bp in length. The SSCP techniques are highly sensitive when combined with polymerase chain reaction. After amplification followed by electrophoresis, mutations in DNA appear as shifts in one or more of the single-stranded DNA bands compared with the wild-type sequence (Fig. 10). Samples are usually run at several temperatures with and without glycerol. DNA AND TISSUE MICROARRAYS DNA microarrays consist of thousands of individual gene sequences bound to regions on the surface of a microscopic glass slide or other support surface (9–13). Arrays are usually a few square centimeters in area and may contain 2000–40,000 or more DNA spots. The complete transcription program of an organism or large segments of the transcription program of normal and physiologically altered or abnormal cells can be

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analyzed rapidly by microarray analysis (9–11). The use of fluorescent labeled cDNA preparations in DNA microarrays allows one to analyze gene expression patterns on a genomic scale. This procedure uses the basic principles of hybridization. A probe hybridizes to multiple defined cDNAs or expressed sequence tags. The probe usually consists of a complex mixture of cDNA fragments derived from mRNA. The probe fragments are usually fluorescent labeled, and they hybridize to the targets on the glass slide. A positive sign is detected by a laser system such as an argon-ion laser and then quantified. Global gene expression can be readily detected with this technique. Another approach is to use oligonucleotide arrays or DNA chips with synthetic oligonucleotides, which represent thousand of gene sequences synthesized on the surface of small areas of a glass slide. DNA array technology for gene expression profiling has been applied to many areas of biomedical sciences. A recent dramatic application is the report by Alizadeh et al. (14), who used gene expression profiling in B-cell lymphomas to identify two molecularly distinct forms of diffuse large B-cell lymphomas that had gene expression patterns indicative of different stages of B-cell differentiation. Another study of gene-expression profiles in hereditary breast cancers found functional differences between breast tumors with BRCA1 mutations and those with BRCA2 mutations (15). Tissue microarrays for high-throughput molecular profiling of normal and tumor specimens have been adopted for rapid screening of many tissues (16,17). This new approach to identify clinically relevant molecular changes uses microarrays from different cases of the same type of tissue or tumor, which can range from 50 to a few hundred sections of tissues on one slide, to check for protein or gene expression in these microsamples. In the near future, a combination of tissue microarray technology with pattern recognition software should greatly advance the discovery of new and potentially useful markers with clinical and diagnostic applications. REFERENCES 1. International Human Genome Sequencing Consortium (2001) Initial sequencing and analysis of the human genome. Nature 409, 860–921. 2. Venter, J.C., Adams, M.D., Myers, E.W., et al. (2001) The sequence of the human genome. Science 291, 1305–1351. 3. Claverie, J.-M. (2001) Gene number. What if there are only 30,000 human genes? Science 291, 1255–1257. 4. Watson, J. D., Hopkins, N. H., Roberts, J. W., Argetsinger Steitz, J., Weiner, A. M. (1987) Molecular Biology of the Gene, 4th ed. Benjamin/Cumming, San Francisco 1987. 5. Lodish, H., Berk, A., Zipinsky, S. L., Matsudaira, P., Baltimore, D., Darnell, J. (2000) Molecular Cell Biology. W. H. Freeman, New York. 6. Hensel, C. H., Xiang, R. H., Sakaguchi, A. Y., Naylor, S. L. (1991) Use of single strand conformation polymorphism technique and PCR to detect p53 gene mutations in small cell lung cancer. Oncogene 6, 1067–1071. 7. Orita, M., Iwahana, H., Kanazawa, H., Hayaski, K., Sekiya, T. (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc. Natl. Acad. Sci. USA 86, 2766–2770. 8. Orita, M., Suzuki, Y., Sekiya, T., Hayashi, K. (1989) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5, 874–879.

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9. Schena, M., Shalon, D., Heller, R., Chai, A., Brown, P. O., Davis, R. W. (1996) Parallel human genome analysis: microarray-based expression monitoring of 1000 genes. Proc. Natl. Acad. Sci. USA 93, 10614–10619. 10. De Risi, J. L., Iyer, V. R., Brown, P. O. (1997) Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278, 680–686. 11. Moch, H., Schraml, P., Bubendorf, L., et al. (1999) High-throughput tissue microarray analysis to evaluate genes uncovered by cDNA microarray screening in renal cell carcinoma. Am. J. Pathol. 154, 981–986. 12. Xu, J., Stolk, J. A., Zhang, X., et al. (2000) Identification of differentially expressed genes in human prostate cancer using subtraction and microarray. Cancer Res. 60, 1677–1682. 13. Kurian, K. M., Watson, C. J., Wyllie, A. H. (1999) DNA chip technology. J. Pathol. 187, 267–271. 14. Alizadeh, A. A., Eisen, M. B., Davis, R. E., et al. (2000) Distinct types of diffuse large Bcell lymphoma identified by gene expression profiling. Nature 403, 503–511. 15. Hedenfalk, I., Duggan, D., Chen, Y., et al. (2001) Gene-expression profiles in hereditary breast cancer. N. Engl. J. Med. 344, 539–548. 16. Kononen, J., Bubendorf, L., Kallioniemi, A., et al. (1998) Tissue microarrays for highthroughput molecular profiling of tumor specimens. Nature Med. 4, 844–847. 17. Moch, H., Schraml, P., Bubendorf, L., et al. (1999) High-throughput tissue microarray analysis to evaluate genes uncovered by cDNA microarray screening in renal cell carcinoma. Am. J. Pathol. 154, 981–986.

2 Laser Capture Microdissection Principles and Applications Ricardo V. Lloyd, MD, PHD INTRODUCTION The acquisition of homogeneous or pure cell populations for cell biologic and molecular analyses has been a difficult challenge for many decades. A variety of approaches have been tried, including macroscopic dissection of tissues from frozen tissue blocks to increase the population of specific cell types (1,2). Some investigators have used irradiation of manually ink-stained sections to destroy cells that were not of interest (3,4). Microdissection with the aid of a microscope and needles has been used by many other investigators (5,6). This latter approach can provide a great deal of precision in collecting homogenous cells, but it is slow and labor-intensive and requires a high degree of manual dexterity. Most of these approaches have not provided the speed, precision, and efficiency needed for research or routine clinical molecular diagnostic use. The recent development of a laser capture microdissection (LCM) unit by the National Cancer Institute (NCI) group led by Liotta and his colleagues (7,8) and by other groups, mainly in Europe (9,10), has provided a rapid and efficient method to capture pure cell populations for molecular and other studies. Principles of LCM Liotta’s group at the NCI first reported on the development of a rapid and reliable method of obtaining homogenous population of cells from complex tissues (7,8). The availability of commercial instruments resulted from a joint venture by the NCI and Arcturus Engineering (Mountain View, CA). With LMC, a complex section of tissues or heterogeneous cell populations on a glass slide is placed on the stage of a specially designed microscope. After the areas or cells of interest are selected, an ethylene vinyl acetate (EVA) transparent film is apposed to the section, and an infrared laser beam is directed at the cell of interest. When the focused laser beam coaxial with the microscope optics is activated, the EVA film above the targeted area melts, surrounds, and holds the cells of interest, which are kept in the film after removal from the glass slide. The film can be placed directly

From: Morphology Methods: Cell and Molecular Biology Techniques Edited by: R. V. Lloyd  Humana Press, Totowa, NJ

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Fig. 1. Laser capture microdissection (LCM) with the Pix Cell instrument. The laser beam is focused on the cut section of cells on the glass slide. The transport arm carries the plastic cap over to align it with the specimen and laser beam. The focused laser beam captures the cells of interest, which adhere to the ethylene vinyl acetate (EVA) film that binds to the captured target cells. After collecting the cells of interest, the transport arm carries the cells back to the side.

into DNA or RNA extraction buffers for nucleic acid extraction and analysis or into other buffers for protein analysis. While in the buffer, the cellular material becomes detached from the film and can be used for cellular or molecular analyses after extraction (see Protocols). Based on numerous reported studies in the literature and our own observations, the LCM procedure does not lead to significant alterations in the morphologic or apparent molecular features of the cells of interest. Technical Aspects The prototypic instrument used for LCM is the Pix Cell developed by Arcturus Engineering and the NCI group (Figs. 1 and 2). The instrument consists of a laser optics deck and illumination tower with a halogen bulb housing, a microscope, and a slide stage with a vacuum chuck. A joystick with XY control and video camera attachment are attached to the microscope. The XY control is for fine positioning of the sample. The laser can be controlled by amplitude and pulse width adjustments using digital controls on the front panel of the electronic box. When the laser fires, an emission indicator is lit, and the power supply emits a beeping sound. Typical operating parameters for the laser for a 30-µm spot is an amplitude of 30 mW and a pulse width of 5 ms; for a 60-µm spot, an amplitude of 50 mW and a pulse width of 5 ms is used. When the operator is ready to capture the cells of interest, a plastic cap is transported with the film carrier and placed on the desired position of the tissue or cells. The laser

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Fig. 2. The cells on the EVA film are placed in the microfuge tube and immersed in the appropriate buffer for DNA, RNA, or protein extraction and analysis.

is then activated, and the EVA film binds to the captured target on the tissue. The laser can be activated as often as is needed to capture the desired numbers of cells. Multiple clusters of homogenous cells can be accumulated into the same polymer EVA film, and individual single laser shots can be used to procure specific cell clusters or individual cells (Fig. 3). In addition, multiple shots can be combined to procure complex but homogenous tissue structures. Up to 3000 shots can be captured on one transfer film cap, which may include up to 6000 cells. Since each shot takes less than 1 s to perform, a large number of cells can be captured in a relatively short period. Technical Variables Technical variables with LCM include the types of analyses that will be done with the tissues relative to fixation and processing, use of frozen versus paraffin tissue sections, and combination of LCM with other techniques such as immunohistochemistry (Immuno-LCM). Tissues fixed in buffered formalin and embedded in paraffin can be used for routine analysis of DNA and in some cases mRNA as well. Tissues are routinely stained with hematoxylin and eosin for optimum visualization of cellular details during LCM. These stains do not affect the integrity of the DNA or RNA. For optimum RNA analysis, frozen tissue sections are preferred. Surprisingly, the nucleic acids are not adversely affected by exposing the tissues to xylene for a short period. RNAse-free conditions are important for obtaining high-quality RNA samples. The section for frozen tissue should be cut at 10 µm or less for optimum transfer, because with thicker sections it is more difficult to visualize single cells. Sections have to be completely dry and not cover-slipped for effective LCM transfer so the final xylene rinse facilitates efficient LCM transfer. After the frozen sections are cut, the slides can be used immediately or stained at −80°C until use. We have observed that

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Fig. 3. LCM capture of cells from normal breast tissue. (A) Groups of normal epithelial cells were selected for LCM (arrowheads). (B) Arrowheads indicate the space from which the cells were captured. (C) Captured cells on the cap with the transfer EVA film. (Reproduced with permission from ref. 13.)

starting with fresh alcohols and xylene for each set of experiments avoids many of the technical problems associated with difficult transfers during LCM. The procedure of combining LCM with immunophenotyping for RNA analysis (immuno-LCM) was first reported from the NCI (11) and subsequently by our groups and others for analyzing single cells (12) and tissues (13–15) (Fig. 4). The critical requirements include using RNAse-free condition and using RNAse inhibitors during the immunostaining procedures. A rapid immunostaining procedure minimizes the time of tissue exposure to RNAse. Various studies have shown that precipitating fixatives such as ethanol and acetone produced better quality reverse transcriptase-polymerase chain reaction (RT-PCR) product amplification compared with crosslinking fixatives such as formaldehyde and glutaraldehyde (11,12,14). However, precipitating fixatives are less effective in some immunostaining procedures compared with crosslinking fixatives (12). Other Laser-Based Microdissection Systems Although LCM is the principal system used, other systems have been developed, mainly in Europe, that are somewhat similar to LCM (16–21). Schutze and Lahr (16)

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Fig. 4. Immuno-laser capture microdissection (LCM). Combined immunohistochemistry and LCM to capture immunophenotyped cells. After ethanol fixing, the tissues are exposed to the specific antibody for about 15 min followed by a streptavidin-biotin-peroxidase reaction and diaminobenzidine (DAB). The immunostained cells (black circles) can be used for LCM and reverse transcriptase-polymerase chain reaction (RT-PCR) and the products analyzed by gel electrophoresis and Southern hybridization.

used a low-power laser to create a gap between the cells of interest and adjacent cells; with an increase in the laser power, they were able to “catapult” the microdissected cells into the cap of a microfuge tube for further analysis without requiring any direct contact with the cells of interest. Using this approach, they could amplify simple cells from archival tissues in the analysis of Ki-ras mutations (16). Fink and colleagues (17) used an ultraviolet light laser to ablate cells that were not of interest and subsequently collected the cells of interest with a stent needle under the control of a micromanipulator (17), which is a modification of the original method of Shibata et al. (3,4). Other investigators have utilized this approach to study microsatellite instability in breast cancer at the single cell level (18). A combination of ultraviolet microbeam microdissection with laser pressure catapulting for the isolation of single chromosomes for development of chromosome-specific paint probes has been reported (19). Becker et al. (20) used a combination of ultraviolet laser microbeams with microdissection and molecular characterization of individual cells to detect a novel mutation in the E cadherin gene in single tumor cells (20). A recent study used the PALM LaserMicrobeam System, which allows the contact-free isolation of single cells or groups of cells using the laser pressure catapulting technique and real-time PCR to study the Her-2/Neu gene and topoisomerase IIα gene in breast cancer specimens (21). Disadvantages of LCM and Related Techniques Although LCM is faster and easier to perform than manual microdissection, there are a few disadvantages. The principal one is that the tissue is not cover-slipped during LCM, so the refractive indices of the dry sections have a refractile quality that obscures

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cellular details at higher magnifications. This can be partially overcome with a diffusion filter on the instrument or by using a drop of xylene on the tissues, which provides wetting and refractive-index matching; the xylene usually evaporates rapidly before microdissection (22). Another disadvantage for investigators with a tight budget is the costs of the instrument. Finally, although LCM is faster than manual methods, a great deal of time is still required to collect cells for an experiment compared with biochemical analysis, especially when thousands of cells are collected (23). APPLICATIONS OF LCM AND RELATED MICRODISSECTION METHODS General Applications The first reports of the development of LCM illustrated the wide applicability of the technique (7,8). The NCI investigators used this technique to analyze loss of heterozygosity (LOH) of the BRCA1 gene in familial breast cancer, chromosome 8p in prostate cancer, the p16 gene in invasive esophageal cancer, and the MEN1 tumor suppressor gene on chromosome 11q13 in gastrinomas (7). Other reported applications using the original carbon dioxide laser included detection of a single base mutation in exon 2 of the von Hippel-Lindau (VHL) gene in hemangioblastoma RT-PCR amplification of actin, prostate-specific antigen, and matrix metalloproteinase-2 in frozen prostate cancer samples and analysis by gelatin zymography, showing the applicability of the LCM technique for enzyme assays (7). Suarea-Quian et al. (24) used an LCM technique with 1-ms laser pulses focus of 6 µm to demonstrated rapid capture of single cells from different types of tissues including immunostained cells (24). Glasow and colleagues (25) utilized LCM to study the leptin receptor in the adrenal by RT-PCR and were able to identify the cell types producing the leptin receptor. In a subsequent study of prolactin receptor (PRL-R) in the adrenal, LCM was used to demonstrate that PRL-R was produced by cortical, but not medullary, cells in the adrenal gland (26). In a detailed analysis of B-cell lymphomas, Fend et al. (27) used LCM to isolate cell populations with different antigen expression patterns. They combined this with PCR and sequencing of clonal immunoglobulin heavy chain rearrangements and clonal rearrangement to show that low-grade B-cell lymphomas with two distinct morphologic and immunophenotypic patterns in the same anatomic site were frequently biclonal (27). This study demonstrates the power of LCM to detect molecular microheterogeneity in complex neoplasms. Our laboratory has used LCM from dissociated cells to study gene expression in individual pituitary cells (12). We have also analyzed normal and tumorous breast tissue by LCM and RT-PCR to show that PRL-R was expressed not only in the epithelial component of normal and tumorous breast tissues but also by stromal cells (13), suggesting modulation of mammary stromal cells by PRL (Figs. 5 and 6). In more recent studies, our laboratory has for the first time obtained pure populations of pituitary folliculo-stellate cells and used these for molecular and cell biologic studies that have provided new insights into the role of these cells in pituitary function (28).

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Fig. 5. RT-PCR and Southern hybridization examination of transforming growth factor-β1 (TGF-β1) and TGF-β-RII with RNA obtained by LCM for normal and neoplastic breast tissues showing representative examples of the analyses. Lanes 1, 3, and 5 are normal breast tissues. Lanes 2, 4, and 6 are invasive carcinoma. Lane 7 represents a positive control breast tissue, and lane 8 is a negative control without reverse transcriptase. Lanes 1–3 and 4–6 represent matching normal and tumor tissues, respectively. Lane 7 is a positive breast tissue control, and lane 8 is a negative control without reverse transcriptase. M, molecular size marker. The top part of each panel represents the RT-PCR results on the gel after ethidium bromide staining. The bottom part is the Southern hybridization with the internal probes. (Reproduced with permission from ref. 13.)

In a recent study of synovial sarcoma, Kasai et al. (29) used LCM and RT-PCR with paraffin wax-embedded tissues to show that the SYT-SSX fusion transcript was present in both the spindle cell and epithelial areas of biphasic synovial sarcomas. This study also reinforced the idea that RT-PCR can be performed on RNA obtained from formalin-fixed, paraffin-embedded tissue sections. In a related study, Shibutani et al.

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Fig. 6. RT-PCR and Southern hybridization examination of prolactin (PRL), prolactin receptor (PRL-R), and GAPDH RNA obtained by LCM showing representative examples of the analyses. Lanes 1, 3, and 5 are normal breast tissues. Lanes 2, 4, and 6 are invasive ductal carcinoma. Lane 7 represents a positive pituitary tissue control, and lane 8 is a negative control without reverse transcriptase. Lanes 1–3 and 4–6 represent matching normal and tumor tissues, respectively. M, molecular size marker. The top part of each panel represents the RT-PCR results on the gel after ethidium bromide staining. The bottom part of each panel is the Southern hybridization with the internal probes. (Reproduced with permission from ref. 13.)

(30) demonstrated that methacarn-fixed, paraffin-embedded tissue could be used for RNA analysis by RT-PCR and for protein analysis by Western blotting after LCM using rat liver as an experimental model. In a study of renal glomerular cells, Kohda et al. (31) used LCM with RT-PCR to detect podoplanin in renal glomerular cells harvested by LCM. They were also able to use LCM to capture pure cell populations from different portions of the tubules, but they could not do this from the collecting ducts.

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In a study of papillary and follicular thyroid carcinoma Gillespie et al. (32) used LCM and loss of heterozygosity (LOH) analysis of highly polymorphic chromosomes to show that LOH was more common in follicular than papillary carcinoma. In a related study on practical clinical applications of LCM, a group of investigators analyzed a primary duodenal carcinoid as well as tumors in the scalp and cervical lymph nodes from the same patient by LCM with 22 markers. The 3p12 marker showed loss in all three tumors, and the 3pl4.2 marker showed an identical shift in the three tumors indicative of a common microsatellite alteration and supporting the notion that the shared molecular abnormalities indicated a common clonal origin with the duodenal carcinoid as the primary tumor (33). Combining LCM with Other Techniques A combination of other techniques with LCM has been reported by various investigators. Jones et al. (34) used LCM and comparative genomic hybridization to study myoepithelial cell carcinomas of the breast and found common alterations such as loss at 16q (3/10 cases) and 17 p (3/10 cases), but they also reported fewer genetic alterations compared with ductal carcinomas of the breast. Shen et al. (35) used LCM and genomewide searching in a study of breast cancers to show that LOH was seen only in ductal carcinoma in situ but not in the invasive component of ductal carcinoma in situ. LCM was advantageous in reducing the heterogeneity within tumors (35). A combination of LCM with fluorescence in situ hybridization (FISH) and flow cytometry was used in a study of normal breast and breast carcinoma from formalin-fixed, paraffin-embedded tissues in which the nuclei were microdissected by LCM (36). Using probes for cyclinD1 and RB1, this group showed amplification of cyclin-D1 of the two cases studied when the results were compared with touch preparations of nuclei from the same fresh tumor specimens. Unfortunately, the numbers of samples analyzed were too small to evaluate, but the preliminary results are promising. DNA Libraries and Arrays Various studies have used LCM to obtain cells as the starting material to clone cDNAs and to analyze genes by cDNA arrays. Several laboratories have used LCM in the initial phase of generating cDNA libraries. The principal leader in this area is the NCI Cancer Genome Anatomy Project (CGAP) (37,38), under which cDNA libraries from human cancers of prostate, ovary, breast, lung, and the gastrointestinal tract will be produced and sequenced. cDNA array technology has been combined with LCM in various studies (39–41). Segroi et al. (40) used LCM and high-density cDNA arrays to study gene expression in purified normal, invasive, and metastatic breast cell populations from a single patient and combined this approach with real-time quantitative PCR and immunohistochemistry to study tumor progression in this tumor model. Leethanakul et al. (41) used LCM and cDNA arrays in studies of squamous cell carcinomas of the head and neck using 5000 cells from normal and tumor tissues. They observed a consistent decrease in cytokeratins and an increase in expression of signal-transducing, cell cycle regulatory proteins, and angiogenic factors, as well as tissue-degrading proteases. Unexpected findings included

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overexpression of the wnt and notch growth and differentiation regulatory systems by these squamous cell carcinomas (41). Proteomic Analysis The use of LCM for protein analysis had been reported in the original descriptions of LCM (7) in which gelatin zymography was used to study gelatinase A (MMP-2) in frozen sections of prostate cancer. In a more recent study, Banks et al. (42) used LCM and two-dimensional electrophoresis to examine protein profiles of selected tissue areas. In the cervix they were able to show enrichment of some proteins compared with the whole tissue using these combined techniques. Emmert-Buck et al. (43) used normal and neoplastic esophageal squamous mucosa as a model to study proteins captured by LCM followed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). They were able to use 50,000 cells to resolve 675 distinct proteins or isoforms with molecular weights between 10 and 200 kDa and isoelectric points between pH 3 and 10. Although 98% of the proteins in normal and tumor samples were identical, 17 of the tumors showed tumor-specific alterations. Seven of these proteins were present only in normal tissues, and 10 of the proteins were present only in the tumors. Cytokeratin I and annexin I were identified as two of the altered proteins. In a study of LCM-captured prostate tissue analyzed with a quantitative chemiluminescent assay, Simone et al. (44) studied prostate-specific antigen (PSA) distribution in normal prostatic intraepithelial neoplasia and carcinoma and found a range of 2 × 104 to 6.3 × 106 PSA molecules per microdissected tissue cells. These observations were concordant with the immunohistochemical staining intensity of tissue sections from these different tissue areas (44). Ornstein et al. (45) used LCM and PAGE (1D and 2D) to analyze PSA α1-antichymotrypsin (ACT) by Western blotting studies and showed a 30-kDa band that was the expected molecular weight of unbound PSA. Binding studies revealed that PSA recovered from LCM-procured cells had the full ability to bind ACT as in the normal prostate. Electrophoretic studies also showed that the PSA/ACT complex was stable, indicating that the complex was similar in normal and tumorous tissues. FUTURE DIRECTIONS Future advances in LCM will include technologic advances in instrumentation and application of LCM to solve difficult and/or challenging problems in cell biology and biomedical sciences. The further development of laser fields small enough to capture single cells or specific parts of cells such as the nucleus are currently under way. Automation of the LCM system for automatic performance of cell capture is also being developed. A major challenge for the technology is to develop methods to capture live cells that can be used for cell culture and other in vitro studies with living cells. Major advances can be anticipated in combining LCM with other techniques such as flow cytometry, FISH and comparative genomic hybridization. Significant advances can be anticipated in gene expression studies with microarrays to profile specific human diseases starting from clearly defined histopathologic cell populations. The technique of “molecular fingerprinting” of entire organs such as the prostate has been initiated by one group (38). Cole et al. (38) have proposed a model for integrating in three

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dimensions the data obtained by LCM and microarrays for the in vivo molecular anatomy analysis of normal and neoplastic cells. These types of approaches will rely heavily on LCM for further advances in understanding the molecular aspects of diseases as they relate to tissue structure and function. REFERENCES 1. Fearon, E. R., Hamilton, S. R., and Vogelstein, B. (1987) Clonal analysis of human colorectal tumors. Science 238, 193–197. 2. Shibata, D., Hawes, D., Li, Z. H., Hernandez, A. M., Spruck, C. H., and Nichols, P. W. (1992) Specific genetic analysis of microscopic tissue after selective ultraviolet radiation fractionation and the polymerase chain reaction. Am. J. Pathol. 141, 539–543. 3. Shibata, D. (1993) Selective ultraviolet radiation fractionation and polymerase chain reaction analysis of genetic alterations. Am. J. Pathol. 143, 1523–1526. 4. Emmert-Buck, M. R., Roth, M. J., Zhuang, Z., et al. (1994) Increased gelatinase A (MMP2) and cathepsin B activity in invasive tumor regions of human colon cancer samples. Am. J. Pathol. 145, 1285–1290. 5. Zhuang, Z., Bertheau, P., Emmert-Buck, M. R., et al. (1995) A microdissection technique for archival DNA analysis of specific cell populations in lesions